This project targets bacterial type IA topoisomerase for discovery of novel antibacterial drugs as countermeasure for multi-drug resistant bacterial pathogens, including gram negatives. Drugs that initiate cell killing by trapping the covalent cleavage complex formed by type IB and type IIA topoisomerases are widely used in current anti-cancer and anti-bacterial therapy. Accumulation of type IA topoisomerase cleavage complex can trigger rapid bacterial cell death via the oxidative cell death pathway but specific inhibitors of type IA bacterial topoisomerase I that can be developed into new antibacterial drugs remain to be discovered. The proposed research activities for the next funding period would develop and utilize rapid cell based applicable for HTS assay format to identify small molecule compounds that have antibacterial activity due to specific interaction with bacterial topoisomerase I. Inhibitors of bacterial topoisomerase I identified from screening would be analyzed biochemically for mechanism of action and structure activity relationship. Priority would be given to compounds with low toxicity that are active against gram negative Escherichia coli and Yersinia pestis in order to identify leads that can be used in development of therapeutic agents against gram negative pathogens. Bacterial proteins that process trapped topoisomerase cleavage complexes will be identified with genetic and biochemical experiments. The repair proteins involved may be useful new targets for combination therapy with antibiotics targeting both type IA and type IIA topoisomerases. Success in these proposed experiments would provide tools and leads for development of novel antibacterial drugs for the urgent public health problem of bacterial pathogens resistant to all current antibiotics.